The aspartyl protease renin is an important modulator of blood pressure. Renin is produced by cleavage of the 395 amino acid zymogen prorenin which circulates in blood at between five and ten times the level of active renin. The putative cleavage site is at the R43L44 sequence of prorenin (Mercure et al., Journal of Biological Chemistry 270(27):16355-16359 (1995)).
Human prorenin is easily activated to renin in vitro with catalytic trypsin. A number of enzymes have been suggested as natural activators of prorenin including the cathepsins, plasmin, and various activated coagulation factors (Mercure et al.).
Once activated, renin hydrolyzes angiotensinogen into angiotensin I. Angiotensin I is further processed into angiotensin II by angiotensin converting enzyme (ACE). Angiotensin II is a potent constrictor of blood vessels which then leads to an elevation of blood pressure. Drugs interfering with the renin-angiotensin system (RAS) are currently being widely developed for the treatment of cardiovascular diseases. They not only lower blood pressure, but also prevent end-organ damage. In an attempt to develop drugs to combat high blood pressure, a number of targets including prorenin, ACE, and renin have been developed.
The only currently prescribed renin therapeutic is the recently introduced direct renin inhibitor aliskiren (Novartis Corporation. Basel, Switzerland). However, due to multiple feedback mechanisms within RAS, RAS blockade, including the inhibition of renin, results in the elevation of both renin and its inactive precursor, prorenin. A rise in renin and prorenin occur particularly following treatment with aliskiren (Novartis). The consequences of such increases in renin and prorenin are currently unknown.
However, it is important to note that prorenin is also elevated in plasma of diabetic subjects before the occurrence of complications with nephropathy and retinopathy. Prorenin, like other components of RAS, can also be detected in urine and may provide several advantages. First, urinary prorenin levels may serve as an early marker for diabetic nephropathy and/or retinopathy and facilitate the selection of patients eligible for treatment with a RAS blocker at a very early stage, Early detection and treatment is pertinent in view of the steep rise in diabetes frequency and the devastating consequences of diabetes in eyes and kidneys. Secondly, changes in plasma and/or urinary prorenin levels might help to monitor the response to RAS blockade.
Nonetheless, there are no commercially available sandwich prorenin ELISA assays. Schalekamp et. al. describe a prorenin ELISA assay using a monoclonal antibody directed against the N-terminal prorenin peptide (Schalekamp et al., Journal of Hypertension 26:928-937 (2008)). This antibody, produced by F. Hoffmann-La Roche AG (Basel, Switzerland), requires extensive and time consuming pretreatment of the prorenin with a renin inhibitor to remove the propiece from the active site, which makes it reactive and unsuitable for common use.
Accordingly, there is a need for prorenin assays without pretreatment. Such prorenin assays would help to identify (diabetic) patients requiring RAS blockade treatment at a very early stage, thereby greatly reducing the occurrence of nephro- and retinopathy. Prorenin measurements may also allow monitoring of the response to RAS blockade, which may help to ascertain why some patients respond well to RAS blockade whereas others do not. Moreover, such measurements would also help to determine the consequences of the changes in (pro)renin concentrations (e.g., (pro)renin receptor activation) that occur during treatment.
Recently, the so-called prorenin receptor was discovered. It is believed that the effects of increased renin and/or prorenin may be exerted via this receptor. Studies suggest that prorenin may function in the absence of cleavage through its binding to the prorenin receptor. Prorenin exists in two conformations: 1) the open conformation, where the active site is accessible, and 2) the closed conformation, where the active site is not accessible. Binding of prorenin to its receptor results in conformation conversion to the open conformation, resulting in non-proteolytic activation. Nguyen et al., J. Clin. Invest. 109:1417 (2002).
Therefore, there is also the need for methods of inhibiting the activation of prorenin. Such methods may include preventing the cleavage of prorenin to form the active renin or preventing prorenin from binding its receptor, such as by the use of an antibody and/or keeping prorenin in its closed conformation.